The development of a non-invasive and reliable assay that reflects the pathogenic process is a highly desired objective in the diagnosis and research of Parkinson's disease (PD). Among the different factors associated with the pathogenic process of PD, α-Syn protein is the most prominent factor.
α-Syn is a presynaptic protein critically involved in the cytopathology and genetics of PD (Lee et al., 2006, Neuron, 52, 33-38). A progressive conversion of the soluble α-Syn protein into insoluble, β-sheet rich filaments and their intraneuronal deposition into Lewy bodies (LB) and Lewy neurites, underlie its cytotoxicity in the synucleinopathies (Duda et al., 2000, NeuroSci Res, 61, 121-127).
Membrane-associated α-Syn has been the focus of many studies. This is primarily because of the findings indicating that upon interactions with membrane phospholipids, the unfolded α-Syn protein acquires an α-helix-rich structure (Davidson et al., 1998, J Biol. Chem, 273, 9443-9449). It is hypothesized that the acquisition of structure is critical for the normal function of the protein. Importantly, the acquisition of structure is implicated in α-Syn aggregation and toxicity (Jo et al., 2000, J. Biol. Chem. 275, 34328-34334). α-Syn interaction with membranes is dependent on the type of lipids (Jo et al., 2000, J. Biol. Chem. 275, 34328-34334), with a preference for anionic head groups and, specifically, phosphatidyl serine (Davidson et al., 1998, J Biol. Chem, 273, 9443-9449). α-Syn interactions with membranes involve the N-terminal region of the protein, consisting of residues 1-95 (Antony B 2011, Annu Rev Biochem, 80, 101-123), which harbors six to seven repeats of the conserved KTKEGV motif (Jakes et al. 1994, FEBS Lett, 345, 27-32). This is consistent with an amphipathic helical domain with the polar face having a net positive charge (Chandra et al., 2003, J Biol Chem. 278, 15313-15318) and the preference for anionic phospholipids (Davidson et al., 1998, J Biol. Chem, 273, 9443-9449).
Altered levels of synuclein proteins have been detected in the CNS of patients with PD and the related synucleinopathies and also in various types of cancer. For example, α-Syn expression is detected in melanoma tumors and nevi (Matsuo et al., 2010, PLoS One, 5(5)-e10481). Members of the synuclein family were shown to be expressed in breast and ovarian cancer cells (Bruening et al., 2002, Cancer, 88, 2154-2163). γ-Syn was found to be overexpressed in ovarian tumors and in ovarian cancer cell lines in contrast to low and almost undetectable levels of γ-syn proteins in the surface epithelial cells of the normal ovary. The expression of γ-syn was seen in 20% of preneoplastic lesions in the ovary, where it showed punctate expression in epithelial inclusion cysts, hyperplastic lesions, and papillary structures.
Furthermore, abnormally high expression of γ-syn has been associated with a wide range of cancer types, including breast, ovarian, cervical, prostrate, liver, pancreatic, colon, gastric, esophagus, and lung compared to almost undetectable levels in adjacent non-neoplastic tissue. In addition to the stage-specific expression of γ-syn, high expression levels in stage III/IV sometimes involving lymph node invasion, was observed in various cancer types. γ-Syn expression is also specifically expressed in high-grade glial tumors such as 33% ependymomas, 63% glioblastomas, and 16% myxopapillary ependymomas, which also demonstrates γ-syn's potential activity as a tumor progression protein is not just restricted to hormone-dependent carcinomas (Ahmad et al. 2007, Faseb J, 21:3419-3430).
Sandwich ELISA methods were recently reported to be a useful diagnostic tool for PD. The sandwich ELISA uses a primary monoclonal anti α-Syn ab for capturing α-Syn antigen from the sample. Then, a general secondary antibody is used for the detection reaction. In addition, a specific sandwich ELISA method was developed to specifically detect oligomeric forms of α-Syn. Specifically, following the initial capturing of α-Syn by the primary monoclonal anti α-Syn ab, the sample is reacted with a polyclonal anti α-Syn ab, that in turn, is used for antigen detection through the HRP-reaction. By using this method it was reported that samples of CSF from PD patients contain a significantly higher ratio of α-Syn oligomers to total α-Syn. In contrast, lower α-Syn levels were found in CSF samples of patients at a progressive state of the disease and drug-naïve patients.
A general disadvantage of the sandwich ELISA method for the detection of α-Syn is that the initial capture step is limited by the specific epitope recognition of the antibody in use. α-Syn undergoes various post translational modifications, including: phosphorylation, ubiquitination, sumoylation, nitrosylation, oligomerization and aggregation. In addition, α-Syn undergoes truncations at its C terminus. Therefore, various α-Syn forms occur in vivo. An additional obstacle is that the physiologic α-Syn forms are yet to be defined and characterized separately from the pathogenic forms of the protein. Therefore, the reliance on a specific antibody, with limited epitope recognition, is a significant disadvantage of this sandwich ELISA method, both with regard to the identification of relevant forms of the protein, and with the level of sensitivity seen with this method.
There is thus a clear need for a highly sensitive assay which can detect and measure α-Syn, even when present at low levels. One purpose of the present invention is to provide such an assay.
A further purpose of the present invention is to use the abovementioned assay for diagnosis of α-Syn-related diseases, determining the severity of said diseases and/or for monitoring a therapeutic regime.
Other aims and purposes will become apparent as the description proceeds.